The present invention relates to a group of genes, and the proteins encoded thereby, which are capable of interfering with Hepatitis B virus (HBV) infection and to methods for identifying, purifying, isolating and characterizing related genes and gene products. The present invention further relates to isolation of soluble forms of the cellular receptor(s) for HBV on hepatocytes from bodily fluids, including, but act limited to, urine, and to purification of these soluble form binding proteins by means including, but not limited to, affinity columns. The present invention further relates to the use of these genes and their translation products to establish experimental models for HBV infection, whether in cell culture or in animals. The present invention further relates to the use of these genes and their translation products for therapeutic purposes. The present invention further relates to the use of these genes and their translation products to screen for additional binding protein interactions. The present invention further relates to the use of these genes and their translation products to prepare specific detectors of these proteins, including, but not limited to, antibodies, phage-display libraries, specific PCR primers, lectin, DNA probes, RNA probes, and non-antibody proteins for diagnostic and therapeutic purposes.
Hepatitis B virus (HBV) is an enveloped RNA virus that infects human liver and replicates via reverse-transcription of the pregenomic RNA Infected patients develop acute hepatitis, which is often self-limiting, but may develop into chronic hepatitis with high risk of liver cirrhosis and primary liver carcinoma in roughly 10% of all cases. The World Health Organization estimates that there will be 400 million carriers Worldwide in year 2000. Effective vaccines exist, but anti viral drugs with good and long term efficacy are not available. Little is known about how HBV infects liver cells and the HBV cellular receptor(s) remain unknown. Many proteins have been identified which bind to the viral envelope associated proteins, HBsAg, or related proteins, but none are considered genuine HBV receptors (reviewed in De et al., 1991 and in references cited therein). Some of these binding proteins are found in serum and some in hepatocytes. None of these molecules have been convincingly tied to infectivity, disqualifying them as genuine HBV receptors. These molecules are of three types, S binding proteins, preS2 binding proteins, and preS1 binding proteins. A brief summary of the characteristics of the three groups is provided herein.
The S binding proteins: HBsAg containing only the S protein binds to a 34-kDa liver protein, which is identified as the phospholipid-binding protein endonexin II (also known as annexin V). Endonexin II has calcium channel activity and it thought to be located primarily, but not exclusively, intracellularly. The biological significance of this remains unclear, as the observed interaction may simply reflect the known ability of endonexin 11 to bind phospholipids, which are abundant in HBsAg lipoprotein. It was subsequently demonstrated that delipidated HBsAg had a drastically diminished capacity to bind endonexin II, leading to speculation that it might play a role in a postbinding membrane fusion event.
It has also been demonstrated that plasma membranes, derived from human liver, contain apolipoprotein H (Apo H), a 46-kDa protein which binds HBsAg. This protein is a glycoprotein with four N-linked carbohydrate chains, which is present in the serum and is not an integral transmembrane protein of the hepatocyte. Its role in infection is uncertain, Moreover, it has been proven that the interaction between Apo H and HBsAg involves triglycerides and not HBV proteins. However, Apo H might play a role in delivery of the virus from the periphery to the liver.
Since binding of these molecules does not involve the preS determinant, they are unlikely to be the sole component of HBV attachment.
The preS2 binding proteins: Some researchers presumed that HBV binds to liver cells via a polymerized form of human serum albumin (pHSA) because a correlation between high viremia and the presence of a so-called pHSA receptor was observed. The preS2-specific domain does possess a pHSA binding activity, however, only pHSA from human or chimpanzee serum binds to preS2. Moreover, pHSA binds to liver cells, albeit in a non-species specific fashion. Furthermore, membranes from fresh human liver are able to bind natural HBs spheres or recombinant preS2 when they art pretreated with pHSA. These observations would suggest that the preS2 domain acts via pHSA as a species- and organ-specific attachment site of HBV except that identification of the exact binding site for pHSA within the preS2 domain is controversial.
The potential importance of pHSA binding for HBV infection has reduced by the observation that native albumin in physiologic concentrations blocks the binding of pHSA to HBsAg. This finding is especially significant considering that the minute concentration of natural pHSA present is serum is negligible when compared with the serum albumin concentration.
The N-linked glycan at the amino end of the preS2 domain has also been suggested as a potential binding site for human hepatocytes on the preS2 domain. This suggestion stems from an unusual glycan structure composed of one mannose chain and two complex chains which is liver specific and able to bind directly to HepG2 cells. Selective removal of this preS2 glycan reduces the preS2 binding by 70%.
It has also been reported that anti-idiotypic antibodies, raised against an epitope localized in the N-terminal part of preS2 protein, recognized human fibronectin, a component of the extracellular matrix. This binding is thought to be species specific because no binding was found between the preS2-associated epitopes with mouse liver. It is currently hypothesized that fibronectin may contribute to the initial binding of the circulating virus.
The preS1 binding proteins: Many researchers suggest possible roles for preS1 binding molecules in viral entry, although no conclusive evidence that these proteins play a role in permissive infection is available.
A portion of preS1, identified as being involved in attachment to HepG2 cells, is highly homologous to the Fc moiety of the xcex1-chain of immunoglobulin A (IgA). Since IgA binds to liver plasma membranes, a common receptor for the attachment of HBV and IgA to human liver cells has bean proposed. However, known receptors for IgA do not appear to be the receptors for HBV.
Anti-idiotypic antibodies have been used to paratope anti-preS(21-47) antibodies, which may represent a mirror image of the binding site on the receptor and thus be able to react with the receptor. These antibodies reacted with a 35-kDa protein and with three other related components of 40-, 43-, and 50-kDa in HepG2 membrane extracts. The 35-kDa protein, identified as the human liver glyceraldehyde-3-phosphate-dehydrogenase (GAPD) is a key enzyme for glycolysis, and the 50-kDa protein seems to contain intrachain disulfide bonds.
In addition, 31-kDa proteins that gross-linked in vitro to a synthetic preS1 peptide (amino acids 21 to 47) has also been identified
Other researchers also identified a 50-kDa protein in normal human serum which interacts with the epitopes localized within the preS1 and preS2 domains. They characterized this molecule as a glycoprotein with N-linked carbohydrate chains, which requires intact disulfide bonds in order to bind preS proteins. This 50-kDa protein blocks the binding of the preS1- and preS2-specific MAbs to HBV. This protein was detected on the surface of human hepatocytes by specific monoclonal antibodies, but not on hepatocytes from other species or in HepG2 cell membranes.
It has also been argued that the asialoglycoprotein receptor on the surface of hepatocytes a responsible for the binding of HBV, mediated by an epitope located in the preS1 domain,
As the expression of the asialoglycoprotein receptor is exclusive to hepatocytes, but not species specific, the presence of HBV in extrahepatic tissue has been explained by the presence of possible asialoglycoprotein-related molecules in these non-hepatic cells.
In summary, although some of the proteins described hereinabove are able is bind virus envelope proteins, they but do not contain the molecular determinants of true receptors. Others with appropriate molecular determinants, fail to bind HBV. None of these molecules have a demonstrable role in initiating HBV infection of hepatocytes.
There is thus a widely recognized need for, and it would be advantageous to identify true HBV binding proteins, which can be effectively used as, for example, therapeutic agents.
While reducing the present invention to practice proteins were purified from concentrated human urine that bind HBsAg preS1 protein and a 29 amino-acids synthetic peptide with the sequence of HBsAg suspected to be essential for HBV infection, that satisfy a possible receptor function. Partial sequence of two of the purified proteins was determined and the corresponding cDNAs were cloned. Interestingly, the two proteins are similar and belong to the same protein family (a third protein was found in an EST library). These three proteins are membrane associated glycoproteins with EGF repeats, a characteristic structure of a very large group of cellular receptor and ligands. One of the proteins (which is referred to herein as UP50) contains also RGD motif that is known to interact with fibronectin and therefore is speculated to be a component of the extracellular matrix. This protein is expressed widely is many tissues but shows highest heel in aorta. Collectively, the data presented herein suggests that these proteins are binding proteins/ligands that may play a role in normal development in general and in HBV infection as cofactors and can therefore be used to modulate virus infection, tissue organization and cell fate and behavior.
Thus, according to one aspect of the present invention there is provided an isolated nucleic acid comprising (a) a polynucleotide at lease 60% identical to SEQ ID NOs:1, 3, 5 or portions thereof as determined using the Bestfit procedure of the DNA sequence analysis software package developed by the Genetic Computer Group (GCG) at the university of Wisconsin (gap creation penaltyxe2x80x9450, gap extension penaltyxe2x80x943); (b) a polynucleotide encoding a polypeptide being at least 60% homologous with SEQ ID NOs:2, 4, 6 or portions thereof as determined using the Bestfit procedure of the DNA sequence analysis software package developed by the Generic computer Group (GCG) at the university of Wisconsin (gap creation penaltyxe2x80x9450, gap extension penaltyxe2x80x943); or (c) a polynucleotide hybridizable with SEQ ID NOs:1, 3, 5 or portions thereof at 68xc2x0 C. in 6xc3x97SSC, 1% SDS, 5xc3x97Denharts, 10% dextran sulfate, 100 xcexcg/ml salmon sperm DNA, and 32p labeled probe and wash at 68xc2x0 C. with 3xc3x97SSC and 0.1% SDS.
According to further features in preferred embodiments of the invention described below, the polynucleotide encodes a polypeptide capable of specifically binding HBV particles.
According to still further features in the described preferred embodiments the polynucleotide encodes a polypeptide capable of specifically binding to HBsAg preS1 protein or a portion thereof.
According to still further features in the described preferred embodiments the polynucleotide encodes a polypeptide capable of specifically binding to a polypeptide as set forth in SEQ ID NOs:8 or 9.
According to still further features in the described preferred embodiments the polynucleotide is as set forth in SEQ ID NOs:1, 3, 5 or portions thereof.
According to another aspect of the present invention there is provided a nucleic acid construct comprising the isolated nucleic acid described herein.
According to yet another aspect of the present invention there is provided a host cell comprising the isolated nucleic acid described herein.
According to still another aspect of the present invention there is provided a transgenic animal comprising the isolated nucleic acid described herein.
According to an additional aspect of the present invention there is provided an antisense molecule capable of base pairing under physiological conditions with a polynucleotide (a) at least 60% identical to SEQ ID NOs:1, 3, 5 or portions thereof as determined using the Bestfit procedure of the DNA sequence analysis software package developed by the Genetic Computer Group (GCG) at the university of Wisconsin (gap creation penaltyxe2x80x9450, gap extension penaltyxe2x80x943); (b) encoding a polypeptide being at least 60% homologous with SEQ ID NOs:2, 4, 5 or portions thereof as determined using the Bestfit procedure of the DNA sequence analysis software package developed by the Genetic Computer Group (GCG) at the university of Wisconsin (gap creation penaltyxe2x80x9450, gap extension penaltyxe2x80x943); or (c) hybridizable with SEQ ID NOs:1, 3, 5 or potions thereof at 68xc2x0 C. in 6xc3x97SSC, 1% SDS, 5xc3x97Denharts, 10% dextran sulfate, 100 xcexcg/ml salmon sperm DNA, and 32p labeled probe and wash at 68xc2x0 C. with 3xc3x97SSC and 0.1% SDS.
According to yet an additional aspect of the present invention there is provided a pharmaceutical composition comprising, as an active ingredient, the antisense molecule described herein, and a pharmaceutically acceptable carrier.
According to still an additional aspect of the present invention there is provided a nucleic acid construct transcribable to produce the antisense molecule described herein.
According to a further aspect of the present invention there is provided a host cell comprising the antisense molecule described herein.
According to yet a further aspect of the present invention there is provided a transgenic animal comprising the antisense molecule described herein.
According to still a further aspect of the present invention there is provided a recombinant protein comprising a polypeptide (a) at least 60% homologous with SEQ ID NOs:2, 4, 6 or portions thereof as determined using the Bestfit procedure of the DNA sequence analysis software package developed by the Genetic Computer Group (GCG) at the university of Wisconsin (gap creation penaltyxe2x80x9450, gap extension penaltyxe2x80x943); (b) encoded by a polynucleotide at least 60% identical to SEQ ID NOs:1, 3, 5 or portions thereof as determined using the Bestfit procedure of the DNA sequence analysis software package developed by the Genetic Computer Group (GCG) at the university of Wisconsin (gap creation penaltyxe2x80x9450, gap extension penaltyxe2x80x943); or (c) encoded by a polynucleotide hybridizable with SEQ ID NOs:3, 5 or portions thereof at 68xc2x0 C. in 6xc3x97SSC, 1% SDS, 5xc3x97Denharts, 10%, dextran sulfate, 100 xcexcg/ml salmon sperm DNA, and 32p labeled probe and wash at 69xc2x0 C. with 3xc3x97SSC and 0.1% SDS.
According to further features in preferred embodiments of the invention described below, the polypeptide is as set fourth in SEQ ID NOs:2, 4, 6 or portions thereof.
According to still further features in the described preferred embodiments the polypeptide is capable of specifically binding HBV particles.
According to still further features in the described preferred embodiments the polypeptide is capable of specifically binding to HBsAg pros l protein or a portion thereof.
According to still further features in the described preferred embodiments the polypeptide is capable of specifically binding to a polypeptide as set forth is SEQ ID NOs:8 or 9.
According to still further features in the described preferred embodiments the recombinant protein is characterized by at least one of the following (a) at least one EGF like domain; (b) at least one transmembrane domain; (c) at least one site for attachment of a hydroxyl side chain; (d) a signal peptide; (e) an RGD attachment sequence; (f) at least one glycosylation site; and (g) at least one disulfide bond.
According to another aspect of the present invention there is provided a pharmaceutical composition comprising, as an active ingredient, the recombinant protein described herein, and a pharmaceutically acceptable carrier.
According to yet another aspect of the present invention there is provided as antibody capable of specific interaction with the recombinant protein described herein.
According to still another aspect of the present invention there is provided a phage display library comprising a plurality of phages each displaying a portion of the recombinant protein described herein.
According to an additional aspect of the present invention there is provided a phage displaying at least a portion of the recombinant protein described herein.
According to yet an additional aspect of the present invention there is provided a method of isolating a polypeptide with HBV binding activity from a biological fluid, the method comprising the steps of (a) producing a purified HBV derived polypeptide; (b) binding the purified HBV derived polypeptide to a solid matrix to thereby obtain an affinity solid matrix; and (c) using the affinity solid matrix for affinity purification of the polypeptide with (HBV) binding activity from the biological fluid.
According to further features in preferred embodiments of the invention described below, the method further comprising the step of concentrating the biological fluid prior to step (c).
According to still further features in the described preferred embodiments the HBV derived polypeptide is e HBV preS1 peptide or a portion thereof.
According to still further features in the described preferred embodiments the HBV derived polypeptide is as set forth in SEQ ID NOs:8 or 9.
According to still further features is the described preferred embodiments the biological fluid is urine.
According to still further features in the described preferred embodiments the biological fluid is concentrated urine.
According to still an additional aspect of the present invention there is provided a method of inhibiting HBV attachment to a hepatic cell the method comprising the step of exposing the cell to a recombinant urine derived protein, the recombinant urine derived protein being capable of binding to a purified HBV derived polypeptide.
According to a further aspect of the present invention there is provided a pharmaceutical composition for inhibiting HBV attachment to a hepatic cell the pharmaceutical composition comprising a recombinant urine derived protein, the recombinant urine derived protein being capable of binding to a purified HBV derived polypeptide, and a pharmaceutically acceptable carrier.
According to yet a further aspect of the present invention there is provided a method of inhibiting HBV attachment to a hepatic cell the method comprising the step of loading the cell with an antisense molecule being targeted against a mRNA encoding a recombinant urine derived protein, the recombinant urine derived protein being capable of binding to a purified HBV derived polypeptide.
According to still a further aspect of the present invention there is provided a pharmaceutical composition for inhibiting HBV attachment to a hepatic cell the pharmaceutical composition comprising an antisense molecule being targeted against a mRNA encoding a recombinant urine derived protein, the recombinant urine derived protein being capable of binding to a purified HBV derived polypeptide, and a pharmaceutically acceptable carrier.
According to further features in preferred embodiments of the invention described below, the purified HBV derived polypeptide is HBsAg preS1 protein or a portion thereof.
According to still further features is the described preferred embodiments the recombinant urine derived protein includes a polypeptide selected from the group consisting of (a) at least 60% homologous with SEQ ID NOs:2, 4, 6 or portions thereof as determined using the Bestfit procedure of the DNA sequence analysis software package developed by the Genetic Computer Group (GCG) at the university of Wisconsin (gap creation penaltyxe2x80x9450, gap extension penaltyxe2x80x943); (b) being encoded by a polynucleotide at least 60% identical to SEQ ID NOs:1, 3, 5 or portions thereof as determined using, the Bestfit procedure of the DNA sequence analysis software package developed by the Genetic Computer Group (GCG) at the university of Wisconsin (gap creations penaltyxe2x80x9450, gap extension penaltyxe2x80x943); and (c) being encoded by a polynucleotide hybridizable with SEQ ID NOs:1, 3, 5 or portions thereof at 68xc2x0 C. in 6xc3x97SSC, 1% SDS, 5xc3x97Denharts, 10% dextran sulfate, 100 xcexcg/ml salmon sperm DNA, 32p labeled probe and wash at 68xc2x0 C. with 3xc3x97SSC and 0.1% SDS.
According to still further features in the described preferred embodiments the polypeptide is as set fourth in SEQ ID NOs:2, 4, 6 or portions thereof.
According to still further features in the described preferred embodiments the polypeptide is capable of specifically binding HBV particles.
According to still further features in the described preferred embodiments the polypeptide is capable of specifically binding to HBsAg preS1 protein or a portion thereof.
According to still further features in the described preferred embodiments the polypeptide is capable of specifically binding to a polypeptide as set forth in SEQ ID NOs:8 or 9.
According to still further features in the described preferred embodiment the recombinant urine derived protein is characterized by at least one of the following (a) at least one EGF like domain; (b) at least one transmembrane domain; (c) at least one site for attachment of a hydroxyl side chain; (d) a signal peptide; (e) an RGD attachment sequence; (f) at least one glycosylation site; and (g) at least one disulfide bond.
The present invention successfully addresses the shortcomings of the presently known configurations by providing new horizons for combating HBV infections and opening new horizons in HBV research.